According to The Breast Cancer Progress Review Group (BC-PRG), an overarching goal for breast cancer etioloogical research is to identify risk factors in the causal pathway of disease that, if changed, will alter the risk of developing breast cancer. One of the recommendations is the study of polymorphisms of genes involved in tumorigenesis, so that subpopulations at increased risk of breast cancer may be identified. In this proposal we plan to investigate the role of mitochondrial DNA (mtDNA) variations a a risk factor for breast cancer. The essential role of mitochondrial oxidative phosphorylation in energy metabolism, the generation of reactive oxygen species, and the initiation of apoptosis have implicated the importance of mitochondria in the process of aging and cancerous growth. Mitochondria are the only animal cellular organelles containing their own genomes that are highly polymorphic among individuals. The DNA variations may affect the stability of the mtDNA, its replication, mtRNA synthesis and processing, the activity of the functional enzymes, and the communication between the nucleus and mitochondria in regulating cell death and growth. In addition, the mitochondrial genome is particularly susceptible to mutation because of the high level of reactive oxygen species (ROS) generated in the organelle environment, coupled with a low level of DNA repair. We hypothesize that mitochondrial DNA variations are modifying risk factors for breast cancer. The polymorphic variations in mtDNA may cause subtle changes in mitochondrial oxidative phosphorylation activity that lead to slightly higher levels of ROS. The accumulation of ROS and oxidative DNA damage during the course of a lifetime may be deleterious. We also hypothesize that somatic mtDNA mutations play a role in tumorigenesis because mutant mitochondria may adapt a replicative advantage leading to abnormal cell growth and ultimately breast cancer. We plan to use the multiplex PCR/ASO (Allele Specific Oligonucleotide) dot blot method to analyze 32 mitochondrial DNA mutations/polymorphisms, known to be associated with DNA damage, tumorigenesis, and mitochondrial disorders, in normal control and breast cancer patients. If the polymorphic mitochondrial DNA variations play a role in modifying breast cancer risk, significant associations of certain mtDNA variations with patients will be observed. We will also screen breast tumors for somatic mutations in the mitochondrial genome by the Temporal Temperature Gradient Gel Electrophoresis (TTGE) method followed by DNA sequencing. If mutant mitochondria play a role in cancerous growth, somatic mtDNA mutations will be detected. Direct identification of mtDNA mutations will generate hypotheses for future research that will elucidate the role of mitochondria in the process of tumorigenesis.